Novel functional assay for proteases and modulators -: Application in β-secretase studies

Proteases play a critical role in many cellular functions and have been an attractive therapeutic target due to their involvement in a number of disease processes. One prominent example is the secretases responsible for the generation of amyloid P peptide, which is believed to be central for the development of Alzheimer's disease. It is therefore desirable to identify and characterize these proteases. We have developed a novel functional approach for identification of proteases and modulators by coupling the protease activity to caspase-mediated apoptosis. Here we show the proof of principle for this approach using beta -secretase as an example. We provide data showing that 1. A modified caspase-3 containing beta -secretase cleavage site induces apoptosis in 293T cells. 2. The modified caspase-3 induced apoptosis is correlated with the susceptibility of beta -secretase recognition sequence to beta -secretase. 3. In vivo beta -secretase competitors BACE2 and BACE2(D110A) prevent the modified caspase-3 induced cell death. Therefore, this approach can be a useful tool in studies of proteolytic cleavage provided only that the protease recognition sequence is known.